Project summary: The existence of long-lived reservoirs of latently infected cells is recognized as a primary barrier to a cure for HIV-1 infection. Thus, the development of therapeutic strategies to eliminate these reservoirs is a critical scientific goal. A key problem in this field is that little is known about the nature of latently infected cells, and the molecular mechanisms that regulate latency. We have recently discovered that latency is associated with a distinct host cell transcriptional signature. Our hypothesis is that this signature represents a host cell transcriptional and epigenetic program that regulates establishment or maintenance of latency. In the set of experiments we propose here, we will test this hypothesis directly, and investigate the molecular mechanisms by which the host cell environment regulates silencing of HIV transcription. This will be achieved directly testing the roles of individual genes from the latency signature in HIV transcription. We will also undertake a detailed analysis of epigenetic pathways that operate in latently infected cells, and directly examine their impact on integrated proviruses. Finally, we will use cutting edge technology that combines time-lapse microscopy and scRNAseq to discover transcriptomic features of cells that correlate with latency reversal. Thus, this dataset will lead to new targets for therapeutic manipulation of latency and guide further experiment to test these hypotheses in in vitro and in vivo models of latency.